The majority of hormones and neurotransmitters communicate information to cells via G protein-coupled receptors (GPCRs), and GPCRs represent the largest group of targets for drug development. The 22 adrenoceptor (22AR) has been one of the most extensively characterized members of the GPCR family. It responds to the catecholamine neurotransmitters epinephrine, norepinephrine and dopamine;and much is known about its agonist binding and G protein coupling domains from extensive mutagenesis studies. The coupling of the 22AR to Gs, the stimulatory protein for adenylyl cyclase, was one of the first hormone activated signaling pathways to be discovered and serves as a paradigm of GPCR signaling. During the past several years the Kobilka lab has made significant progress towards characterizing the structural changes associated with agonist activation, and has recently obtained a crystal structure of the wild type 22AR in complex with a Fab fragment, as well as a crystal structure of a 22AR that has been modified to improve its structural stability. The Sunahara lab recently developed recombinant HDL phospholipid particles as an ideal biochemical system for studying structural interactions between the 22AR and Gs. This proposal represents a close collaboration that combines the expertise of these two labs to characterize the structural interactions between the 22AR and Gs. The findings will likely apply to the large number of closely related monoamine receptors and to GPCRs in general. Moreover, the methodologies developed for characterizing 22AR coupling to Gs will be applicable to other GPCRs. A better understanding of the structure and mechanism of activation of the 22AR-Gs complex will further the potential for structure-based drug design and in silico screening for GPCR targets, leading to more rapid development of highly selective and effective drugs. the specific aims are: aim 1. characterize the structural dynamics of 22ar coupling to Gs. We will use biophysical approaches to examine the structural basis of functional cooperativity observed in the 22AR-Gs complex. We will examine the effect of Gs on the structure of the 22AR, and the effect of the 22AR on the structure of Gs. We will examine coupling of Gs to 22AR monomers and oligomers. In addition, we will study the effects of ligands having different efficacies on interactions between the 22AR and Gs. Aim 2. Determine the structure of the 22ar-gs complex. We will take several complementary approaches to obtain a high-resolution crystal structure of the 22AR-Gs complex. We will also use single particle imaging by cryoelectron microscopy to study the structure of the 22AR-Gs complex in a native lipid environment. Public Health Relevance: The goal of this proposal is to determine the mechanism by which G protein coupled receptors (GPCRs) respond to hormones and neurotransmitters, and modify the function of cells. This information will facilitate the process of drug discovery for GPCRs, which are the largest family of membrane proteins in the human genome. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease.